Development and validation of an integrative pan-solid tumor predictor of PD-1/PD-L1 blockade benefit.

BACKGROUND: Anti-PD-1 and PD-L1 (collectively PD-[L]1) therapies are approved for many advanced solid tumors. Biomarkers beyond PD-L1 immunohistochemistry, microsatellite instability, and tumor mutation burden (TMB) may improve benefit prediction. METHODS: Using treatment data and genomic and transcriptomic tumor tissue profiling from an observational trial (NCT03061305), we developed Immunotherapy Response Score (IRS), a pan-tumor predictive model of PD-(L)1 benefit. IRS real-world progression free survival (rwPFS) and overall survival (OS) prediction was validated in an independent cohort of trial patients. RESULTS: Here, by Cox modeling, we develop IRS-which combines TMB with CD274, PDCD1, ADAM12 and TOP2A quantitative expression-to predict pembrolizumab rwPFS (648 patients; 26 tumor types; IRS-High or -Low groups). In the 248 patient validation cohort (248 patients; 24 tumor types; non-pembrolizumab PD-[L]1 monotherapy treatment), median rwPFS and OS are significantly longer in IRS-High vs. IRS-Low patients (rwPFS adjusted hazard ratio [aHR] 0.52, p = 0.003; OS aHR 0.49, p = 0.005); TMB alone does not significantly predict PD-(L)1 rwPFS nor OS. In 146 patients treated with systemic therapy prior to pembrolizumab monotherapy, pembrolizumab rwPFS is only significantly longer than immediately preceding therapy rwPFS in IRS-High patients (interaction test p = 0.001). In propensity matched lung cancer patients treated with first-line pembrolizumab monotherapy or pembrolizumab+chemotherapy, monotherapy rwPFS is significantly shorter in IRS-Low patients, but is not significantly different in IRS-High patients. Across 24,463 molecularly-evaluable trial patients, 7.6% of patients outside of monotherapy PD-(L)1 approved tumor types are IRS-High/TMB-Low. CONCLUSIONS: The validated, predictive, pan-tumor IRS model can expand PD-(L)1 monotherapy benefit outside currently approved indications.

Therapies activating the immune system (checkpoint inhibitors) have revolutionized the treatment of patients with advanced cancer, however new molecular tests may better identify patients who could benefit. Using treatment data and clinical molecular test results, we report the development and validation of Immunotherapy Response Score (IRS) to predict checkpoint inhibitor benefit. Across patients with more than 20 advanced cancer types, IRS better predicted checkpoint inhibitor benefit than currently available tests. Data from >20,000 patients showed that IRS identifies ~8% of patients with advanced cancer who may dramatically benefit from checkpoint inhibitors but would not receive them today based on currently available tests. Our approach may help clinicians to decide which patients should receive checkpoint inhibitors to treat their disease.

Ocular adverse events associated with chimeric antigen receptor T-cell therapy: a case series and review.

BACKGROUND/AIMS: Chimeric antigen receptor T-cell (CAR T) therapy has been shown to improve the remission rate and survival for patients with refractory haematological malignancies. The aim of this study is to describe ocular adverse effects associated with CAR T therapy in patients with haematological malignancies. METHODS: This is a retrospective, single-institution, case series. Patients aged 18 years or older who received standard of care CAR T therapy for relapsed/refractory large B-cell lymphoma with a documented ophthalmic evaluation were included. The primary outcome was clinician ophthalmic examination findings. RESULTS: A total of 66 patients received CAR T-cell therapy from February 2018 to October 2019 with 11 receiving an ophthalmic examination. Eleven patients (n=22 eyes) who received CAR T-cell therapy were included in review. The median time from CAR T-cell infusion date to ocular examination was 57.5 days. The median patient age at the time of examination was 60.5 years. Ten patients had subjective symptoms prompting ophthalmic examination. Two patients reported floaters and photopsias. One patient had worsening ocular graft-versus-host disease. Two patients were identified with possible reactivation of viral infections, including herpes zoster ophthalmicus and regressing acute retinal necrosis. CONCLUSIONS: The increasing use of CAR T therapy for malignancies underscores the importance of ophthalmologists and oncologists understanding the potential toxicities associated with its use, particularly ocular toxicities and when to refer for an ophthalmic examination.

Liquid-based rapid onsite evaluation of endobronchial ultrasound cytologies.

INTRODUCTION: Rapid onsite evaluation (ROSE) generally uses smears made at the site of the procedure ("smear-based ROSE"). It requires considerable time, generally 2 individuals, technical expertise, and it can be difficult to estimate material available for ancillary studies. We developed an alternative ROSE using liquid-based cytology ThinPrep with hematoxylin and eosin (H&E) stain ("liquid-based ROSE") and assessed its advantages. MATERIALS AND METHODS: Clinicians rinse the sample(s) into CytoRich Red and send to Pathology. A defined proportion of the needle rinse is removed for a ThinPrep stained with a rapid H&E. Adequacy and diagnosis were compared to final outcome. Total time was recorded. RESULTS: Among 52 liquid-based ROSE readings, 28 (53.8%) were interpreted as "adequate" with final as adequate; 17 (32.7%) were interpreted as "inadequate" with final as inadequate; 7 (13.5%) were interpreted as "inadequate" with final as adequate. Of 23 readings provided with onsite diagnosis, 15 (65.2%) were interpreted as definitive positive or negative diagnoses; 6 (26%) were interpreted as nondiagnostic; and 2 (8.7%) were interpreted as atypical. All definitive diagnoses were concordant with final diagnoses. The time for liquid ROSE performance ranges from 6 to 22 minutes (mean: 13 minutes) and required only 1 individual. CONCLUSIONS: Liquid-based ROSE allows accurate adequacy determination and diagnosis, takes about 15 minutes of cytologist time, and can be performed by just 1 person. The technique produces well-preserved and stained slides, it may allow a better estimation of the total amount of material in the specimen vial and may provide a better platform for telecytology.

Fluorescence Polarization Imaging of Methylene Blue Facilitates Quantitative Detection of Thyroid Cancer in Single Cells.

BACKGROUND: Diagnostic accuracy of the standard of care fine-needle aspiration cytology (FNAC) remains a significant problem in thyroid oncology. Therefore, a robust and accurate method for reducing uncertainty of cytopathological evaluation would be invaluable. METHODS: In this double-blind study, we employed fluorescence emission and quantitative fluorescence polarization (Fpol) confocal imaging for sorting thyroid cells into benign/malignant categories. Samples were collected from malignant tumors, benign nodules, and normal thyroid epithelial tissues. RESULTS: A total of 32 samples, including 12 from cytologically indeterminate categories, were stained using aqueous methylene blue (MB) solution, imaged, and analyzed. Fluorescence emission images yielded diagnostically relevant information on cytomorphology. Significantly higher MB Fpol was measured in thyroid cancer as compared to benign and normal cells. The results obtained from 12 indeterminate samples revealed that MB Fpol accurately differentiated benign and malignant thyroid nodules. CONCLUSIONS: The developed imaging approach holds the potential to provide an accurate and objective biomarker for thyroid cancer, improve diagnostic accuracy of cytopathology, and decrease the number of lobectomy and near-total thyroidectomy procedures.

Deconvolution microscopy: A platform for rapid on-site evaluation of fine needle aspiration specimens that enables recovery of the sample.

CONTEXT: Rapid on-site evaluation (ROSE) optimises the performance of cytology, but requires skilled handling, and smearing can make the material unavailable for some ancillary tests. There is a need to facilitate ROSE without sacrificing part of the sample. OBJECTIVE: We evaluated the image quality of inexpensive deconvolution fluorescence microscopy for optically sectioning non-smeared fine needle aspiration (FNA) tissue fragments. DESIGN: A portion of residual material from 14 FNA samples was stained for 3 min in Hoechst 33342 and Sypro™ Red to label DNA and protein respectively, transferred to an imaging chamber, and imaged at 200× or 400× magnification at 1 micron intervals using a GE DeltaVision inverted fluorescence microscope. A deconvolution algorithm was applied to remove out-of-plane signal, and the resulting images were inverted and pseudocoloured to resemble H&E sections. Five cytopathologists blindly diagnosed 2 to 4 representative image stacks per case (total 70 evaluations), and later compared them to conventional epifluorescent images. RESULTS: Accurate definitive diagnoses were rendered in 45 (64%) of 70 total evaluations; equivocal diagnoses (atypical or suspicious) were made in 21 (30%) of the 70. There were two false positive and two false negative "definite" diagnoses in three cases (4/70; 6%). Cytopathologists preferred deconvolved images compared to raw images (P < 0.01). The imaged fragments were recovered and prepared into a ThinPrep or cell block without discernible alteration. CONCLUSIONS: Deconvolution improves image quality of FNA fragments compared to epifluorescence, often allowing definitive diagnosis while enabling the ROSE material to be subsequently triaged.

Clinical, pathologic, and molecular analyses of superficial low-grade fibromyxoid sarcoma in two young patients: A rare and deceptive mimic of benignancy.

Low-grade fibromyxoid sarcoma (LGFMS) is a histopathologically deceptive soft tissue neoplasm with bland cytology, which is typically encountered in deep soft tissue of adults. We report two cases of superficial LGFMS in young patients (16 and 21 years old, respectively), which were difficult to diagnose on histopathologic and clinical findings alone. LGFMS commonly mimics benign neoplasms such as cellular neurothekeoma, fibromatosis, neurofibroma, and perineurioma. Malignancies included in the differential diagnosis are soft tissue neoplasms such as dermatofibrosarcoma protuberans and myxofibrosarcoma. A high degree of reported variation in pattern and cellularity among LGFMS further complicates the diagnosis. Careful examination and appropriate immunohistochemistry panels including MUC4 are essential for narrowing the differential diagnosis. Molecular studies for possible FUS translocation can confirm the diagnosis of LGFMS. Sufficient sampling and workup of these lesions are critical, especially in younger patients. Young age and superficial presentation can easily sway dermatopathologists/dermatologists toward an incorrect diagnosis of benignancy.

Angiokeratoma-like purpuric palmar nodules following chemotherapy.

We describe a patient with leukemia undergoing chemotherapy who developed painful purpuric nodules of the digits. These findings were concerning for endocarditis (clinically) and angiokeratomas on gross histology. After extensive evaluation, we report the development of painful purpuric nodules as a likely side effect of the patient's therapeutic regimen (hydroxyurea, danorubicin, cytarabine, and methotrexate).

Real-World Performance of a Comprehensive Genomic Profiling Test Optimized for Small Tumor Samples.

PURPOSE: Tissue-based comprehensive genomic profiling (CGP) is increasingly used for treatment selection in patients with advanced cancer; however, tissue availability may limit widespread implementation. Here, we established real-world CGP tissue availability and assessed CGP performance on consecutively received samples. MATERIALS AND METHODS: We conducted a post hoc, nonprespecified analysis of 32,048 consecutive tumor tissue samples received for StrataNGS, a multiplex polymerase chain reaction (PCR)-based comprehensive genomic profiling (PCR-CGP) test, as part of an ongoing observational trial (NCT03061305). Sample characteristics and PCR-CGP performance were assessed across all tested samples, including exception samples not meeting minimum input quality control (QC) requirements (< 20% tumor content [TC], < 2 mm2 tumor surface area [TSA], DNA or RNA yield < 1 ng/µL, or specimen age > 5 years). Tests reporting ≥ 1 prioritized alteration or meeting TC and sequencing QC were considered successful. For prostate carcinoma and lung adenocarcinoma, tests reporting ≥ 1 actionable or informative alteration or meeting TC and sequencing QC were considered actionable. RESULTS: Among 31,165 (97.2%) samples where PCR-CGP was attempted, 10.7% had < 20% TC and 59.2% were small (< 25 mm2 tumor surface area). Of 31,101 samples evaluable for input requirements, 8,089 (26.0%) were exceptions not meeting requirements. However, 94.2% of the 31,101 tested samples were successfully reported, including 80.5% of exception samples. Positive predictive value of PCR-CGP for ERBB2 amplification in exceptions and/or sequencing QC-failure breast cancer samples was 96.7%. Importantly, 84.0% of tested prostate carcinomas and 87.9% of lung adenocarcinomas yielded results informing treatment selection. CONCLUSION: Most real-world tissue samples from patients with advanced cancer desiring CGP are limited, requiring optimized CGP approaches to produce meaningful results. An optimized PCR-CGP test, coupled with an inclusive exception testing policy, delivered reportable results for > 94% of samples, potentially expanding the proportion of CGP-testable patients and impact of biomarker-guided therapies.

Development and Validation of StrataNGS, a Multiplex PCR, Semiconductor Sequencing-Based Comprehensive Genomic Profiling Test.

Despite widespread use in targeted tumor testing, multiplex PCR/semiconductor (Ion Torrent) sequencing-based assessment of all comprehensive genomic profiling (CGP) variant classes has been limited. Herein, we describe the development and validation of StrataNGS, a 429-gene, multiplex PCR/semiconductor sequencing-based CGP laboratory-developed test performed on co-isolated DNA and RNA from formalin-fixed, paraffin-embedded tumor specimens with ≥2 mm2 tumor surface area. Validation was performed in accordance with MolDX CGP validation guidelines using 1986 clinical formalin-fixed, paraffin-embedded samples and an in-house developed optimized bioinformatics pipeline. Across CGP variant classes, accuracy ranged from 0.945 for tumor mutational burden (TMB) status to >0.999 for mutations and gene fusions, positive predictive value ranged from 0.915 for TMB status to 1.00 for gene fusions, and reproducibility ranged from 0.998 for copy number alterations to 1.00 for splice variants and insertions/deletions. StrataNGS TMB estimates were highly correlated to those from whole exome- or FoundationOne CDx-determined TMB (Pearson r = 0.998 and 0.960, respectively); TMB reproducibility was 0.996 (concordance correlation coefficient). Limit of detection for all variant classes was <20% tumor content. Together, we demonstrate that multiplex PCR/semiconductor sequencing-based tumor tissue CGP is feasible using optimized bioinformatic approaches described herein.

Lenvatinib Targets PDGFR-ß Pericytes and Inhibits Synergy With Thyroid Carcinoma Cells: Novel Translational Insights.

CONTEXT: Pericyte populations abundantly express tyrosine kinases (eg, platelet-derived growth factor receptor-ß [PDGFR-ß]) and impact therapeutic response. Lenvatinib is a clinically available tyrosine kinase inhibitor that also targets PDGFR-ß. Duration of therapeutic response was shorter in patients with greater disease burden and metastasis. Patients may develop drug resistance and tumor progression. OBJECTIVES: Develop a gene signature of pericyte abundance to assess with tumor aggressiveness and determine both the response of thyroid-derived pericytes to lenvatinib and their synergies with thyroid carcinoma-derived cells. DESIGN: Using a new gene signature, we estimated the relative abundance of pericytes in papillary thyroid carcinoma (PTC) and normal thyroid (NT) TCGA samples. We also cocultured CD90+;PAX8- thyroid-derived pericytes and BRAFWT/V600E-PTC-derived cells to determine effects of coculture on paracrine communications and lenvatinib response. RESULTS: Pericyte abundance is significantly higher in BRAFV600E-PTC with hTERT mutations and copy number alterations compared with NT or BRAFWT-PTC samples, even when data are corrected for clinical-pathologic confounders. We have identified upregulated pathways important for tumor survival, immunomodulation, RNA transcription, cell-cycle regulation, and cholesterol metabolism. Pericyte growth is significantly increased by platelet-derived growth factor-BB, which activates phospho(p)-PDGFR-ß, pERK1/2, and pAKT. Lenvatinib strongly inhibits pericyte viability by down-regulating MAPK, pAKT, and p-p70S6-kinase downstream PDGFR-ß. Critically, lenvatinib significantly induces higher BRAFWT/V600E-PTC cell death when cocultured with pericytes, as a result of pericyte targeting via PDGFR-ß. CONCLUSIONS: This is the first thyroid-specific model of lenvatinib therapeutic efficacy against pericyte viability, which disadvantages BRAFWT/V600E-PTC growth. Assessing pericyte abundance in patients with PTC could be essential to selection rationales for appropriate targeted therapy with lenvatinib.

Removal of equine cryptorchid testes through an enlarged umbilical portal in dorsally recumbent horses after intra-abdominal laparoscopic castration.

BACKGROUND: Laparoscopic removal of cryptorchid testes has been routinely reported through enlarged parainguinal incisions in dorsally recumbent horses. Outcomes following removal through an extended umbilical incision have not been previously reported. OBJECTIVE: To describe the surgical technique of removing cryptorchid testes in dorsally recumbent horses through an enlarged umbilical portal after laparoscopic intra-abdominal castration. STUDY DESIGN: Retrospective case series. METHODS: Medical records were reviewed for horses that underwent laparoscopic removal of unilateral or bilateral cryptorchid testes from January 2006 to December 2016. Horses were placed under general anaesthesia, positioned in dorsal recumbency and then tilted into Trendelenburg position. Cryptorchid testes were castrated by ligating loop application and/or electrosurgery. The umbilical portal incision was extended along the linea alba for testes removal. All descended testes were removed by routine closed castration with the scrotal incision left to heal by second intention. Perianaesthetic laboratory values, surgical procedure descriptions, surgery and anaesthesia times, and in-hospital perioperative complications were recorded. RESULTS: A total of 79 horses, aged 1-14 years, with unilateral or bilateral cryptorchidism were included: 90 cryptorchid testes were successfully removed through an enlarged umbilical portal incision. Sixty-eight horses were unilaterally cryptorchid and 11 were bilaterally cryptorchid. Two horses had minor post-operative complications related to the extended umbilical portal incision. MAIN LIMITATIONS: Retrospective nature of the study, no control population for comparison and no follow-up after hospital discharge. CONCLUSION: An extended umbilical portal incision is a successful alternative to extending a parainguinal incision for testis removal after laparoscopic castration.

Updates on the Pathology and Management of Nail Unit Tumors and Dermatoses.

Nail unit pathology is indispensable to reach an accurate diagnosis of nail tumors as well as inflammatory disorders. This review article provides an update from the most recently published studies on the pathology and management of nail unit tumors and inflammatory disorders. Recent findings of nail clipping histopathology are described first, followed by discussing recent data on the diagnosis and surgical management of several types of nail unit tumors, ending with discussing the recent discoveries in selected nail unit inflammatory disorders.

Diagnostic value of a comprehensive, urothelial carcinoma-specific next-generation sequencing panel in urine cytology and bladder tumor specimens.

BACKGROUND: Urine cytology can reliably diagnose high-grade urothelial carcinoma (HGUC) but not low-grade urothelial carcinoma (LGUC), and a more sensitive test is needed. Previously, a pilot study highlighted the possible diagnostic utility of next-generation sequencing (NGS) in identifying both LGUC and HGUC in urine cytology specimens. METHODS: Twenty-eight urine ThinPrep cytology specimens and preceding or subsequent bladder tumor biopsy/resection specimens obtained within 3 months were included in the study (LGUC, n = 15; HGUC, n = 13). A customized, bladder-specific NGS panel was performed; it covered 69 frequently mutated or altered genes in urothelial carcinoma (UC) that were reported by The Cancer Genome Atlas and the Catalogue of Somatic Mutations in Cancer. RESULTS: The sequencing results were compared between the urine cytology specimens and the corresponding bladder tumor biopsies/resections. TP53 was the most frequently identified mutation in HGUC cases (11 of 13 [85%]). PIK3CA and KDM6A were the most frequently identified mutations in LGUC: they occurred in 7 of 15 cases (47%) and in 6 of 15 cases (40%), respectively. Additional frequent mutations identified in the panel included ARID1A (n = 5), EP300 (n = 4), LRP1B (n = 3), ERBB2 (n = 2), STAG2 (n = 2), FGFR3 (n = 3), MLL (n = 2), MLL3 (n = 2), CREBBP1 (n = 1), RB1 (n = 1), and FAT4 (n = 1). Overall, the concordance between the cytology and surgical specimens was 75%. The sensitivity and specificity for identifying mutations in urine cytology specimens were 84% and 100%, respectively. CONCLUSIONS: A bladder-specific NGS panel increases the sensitivity and specificity of urine cytology's diagnostic utility in both low- and high-grade tumors and may serve as a noninvasive surveillance method in the follow-up of patients with UC harboring known mutations.

Colposcopic endocervical brushing cytology appears to be more sensitive than histologic endocervical curettage for detecting endocervical adenocarcinoma.

INTRODUCTION: Colposcopic endocervical brushing cytology (CEB) is more sensitive than endocervical curettage (ECC) for detecting squamous intraepithelial lesions. There are no data on performance of CEB for detecting endocervical adenocarcinoma. MATERIALS AND METHODS: A total of 151 patients were identified in a word search for "endocervical adenocarcinoma" in surgical pathology reports from January 2007 to June 2019. To measure sensitivity, reports of CEB or ECC samples within 1 year preceding the first surgical pathology diagnosis of at least endocervical adenocarcinoma in situ (AIS+) were examined. Specificity was measured in a cohort in which at least atypical glandular cells (AGC+) was reported in CEB or ECC. RESULTS: Seven CEB preceding diagnosis of AIS were identified: 6 of 7 were positive or suspicious for AIS+. One of 7 was negative and it was negative on re-review. Three of 6 positive CEB cases used cell blocks with immunohistochemistry. Seventy ECC samples preceding diagnosis of AIS were identified: 40 of 70 were diagnosed as AGC+. The sensitivities of CEB and ECC for detecting AIS+ at a threshold of AGC+ are 86% and 57% (too few patients for statistics), respectively. For specificity, 12 of 18 CEB and 9 of 25 ECC reports with AGC+ were false positive by follow-up surgical pathology. The specificities of CEB and ECC are 99.4% and 99.9%, respectively. CONCLUSION: Sensitivity of CEB for detecting AIS+ (86%) is at least as high as ECC (57%). Specificity of CEB is similar to ECC. Addition of a cell block to CEB may be useful. CEB appears to be an appropriate test for follow-up of atypical glandular cells reported on Papanicolaou tests.

The Utility of MYB Immunohistochemistry (IHC) in Fine Needle Aspiration (FNA) Diagnosis of Adenoid Cystic Carcinoma (AdCC).

Differentiating adenoid cystic carcinoma (AdCC) from other basaloid neoplasm in a fine needle aspiration (FNA) sample can be challenging. Activation of MYB in AdCC by the fusion transcript MYB-NFIB has been recently demonstrated in salivary gland and other organs. The aim of this study is to evaluate the utility of MYB immunohistochemistry (IHC) in distinguishing AdCCs and other basaloid neoplasm in cytology specimens. Eighteen FNA cases, from salivary gland and other sites, and their subsequent surgical resection specimens were included in the study. Eight cases were confirmed AdCC on resection. MYB IHC was performed on slides made from cytology cell block and surgical resection paraffin blocks. Percentage and intensity of nuclear staining in tumor cells was scored as 0 to 3. The staining results were concordant between cytology specimens and their corresponding surgical resection tumors. Strong diffuse nuclear staining (score 3, N = 5) was exclusively observed in AdCC, both in cytology and surgical specimens. Only one pleomorphic adenoma and one poorly differentiated basaloid carcinoma were positive for MYB staining (score 1 to 2). Any degree of nuclear MYB labeling was seen in 100% AdCC cases (N = 8/8) compared with of 20% (N = 2/10) of all other non-AdCC cases (P = < 0.001). The sensitivity and specificity of any degree MYB positivity for AdCC in cytology specimen is 100% and 78%. The sensitivity and specificity of strong diffuse MYB labeling (score 2 to 3) for AdCC is 83% and 100% in cytology specimen. Strong diffuse nuclear staining of MYB is valuable in supporting a cytologic diagnosis of AdCC. However, weak and focal labeling of MYB should be interpreted with caution as it can be seen in benign and other malignant basaloid lesions.

Shapiro xanthogranuloma: An essential diagnosis for dermatologists and dermatopathologists to recognize to avoid misdiagnosis of a hematopoietic malignancy in infants and neonates.

The Shapiro xanthogranuloma is a histopathologic form of xanthogranuloma that shows closely packed monomorphous cells, which can extend into the subcutaneous fat; it usually lacks routine diagnostic features of xanthogranuloma. Herein we describe two cases of Shapiro xanthogranuloma occurring in a neonate and in an infant, which were initially thought to be hematologic malignancies. One patient's presentation as a "blueberry muffin baby" added to the diagnostic confusion. Pediatric dermatologists, dermatologists, and dermatopathologists need to be aware of the Shapiro xanthogranuloma and its clinicopathologic features to avoid misdiagnosis of a hematopoietic malignancy in neonates and infants.